- Email: [email protected]
The 188Re(III)–EDTA complex—a multipurpose starting material for the preparation of relevant 188Re complexes under mild conditions
ARTICLE IN PRESS
Applied Radiation and Isotopes 64 (2006) 223–227 www.elsevier.com/locate/apradiso
The 188Re(III)–EDTA complex—a multipurpose starting material for the preparation of relevant 188Re complexes under mild conditions S. Seifert, H.-J. Pietzsch Forschungszentrum Rossendorf, Institut fu¨r Bioanorganische und Radiopharmazeutische Chemie, Postfach 510 119, D-01314 Dresden, Germany Received 25 July 2005; received in revised form 17 August 2005; accepted 20 August 2005
Abstract An easy and gentle method for the preparation of 188Re(V) complexes with bidentate and tetradentate ligands is described starting from the precursor complex 188Re(III)–EDTA. That complex is prepared at room temperature in acidic solution and reacts by a combined re-oxidation/ligand exchange reaction with appropriate ligands like DMSA or ECD (DMSA ¼ dimercapto succinic acid, ECD ¼ L,L-ethylene dicysteine diethyl ester) or en, tau, and cyclam (en ¼ ethylene diamine, tau ¼ 1,4,8,11-tetraazaundecane, cyclam ¼ 1,4,8,11-tetraazacyclo-tetradecane) to the 188 Re(V)-oxo- and dioxocomplexes, respectively. The chelates were unambiguously identified by chromatographic comparison with spectroscopically characterised samples or known 99mTc-kit reconstitutions. The reaction succeeds under mild conditions (room temperature, short time, neutral or weak basic solutions) with high yields and has potential for labelling of sensitive biomolecules with 188Re. r 2005 Elsevier Ltd. All rights reserved. Keywords:
188
Re(V) complexes; Ligands; Re-oxidation
1. Introduction Rhenium-188 belongs to the most favourable radionuclides offering potential for targeted radiotherapy of cancer. It is currently obtained from the parent nuclide tungsten-188 through a generator system (Knapp et al., 1994). Due to its easy availability and suitable nuclear properties (Ebmax ¼ 2.1 MeV, t1/2 ¼ 16.9 h), 188Re is an attractive isotope for developing therapeutic radiopharmaceuticals (Bla¨uenstein, 1990; Blower et al., 1996; Prakash et al., 1996; Lin et al., 1999). The Corresponding author. Tel.: +49 351 2602442; fax: +49 351 2603232. E-mail address: [email protected] (S. Seifert).
associated g-emission (Eg ¼ 155 keV) could be additionally utilised for imaging and dosimetric purposes. Generally, there is a wide chemical analogy between 188 Re and 99mTc. Differences are observed in the kinetics of ligand exchange reactions and the redox behaviour. Especially the reduction of the perrhenate generator eluate in various ligand solutions needs more drastic conditions than those used for pertechnetate eluate. Normally, milligram amounts of stannous chloride or other reducing agents have to be used for complete reduction, e. g., of perrhenate to Re(V)-oxocomplexes with ‘‘N2S2’’- or ‘‘N3S’’-ligands, in contrast to the microgram amounts, necessary for the reduction of pertechnetate (Dadachova and Mirzadeh, 1997; Oh et al., 2003). Moreover, the complex formation only
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S. Seifert, H.-J. Pietzsch / Applied Radiation and Isotopes 64 (2006) 223–227
succeeds in limited volumes and in the majority of cases at high temperatures and under strongly acidic conditions (Mitterhauser et al., 2004; Hsieh et al., 1999). All these facts restrain the labelling of sensitive biomolecules according to the bifunctional approach. Besides the low valent organometallic tricarbonyl complexes (Alberto et al., 1999; Schibli et al., 2002) and the ‘4+1’ complexes at oxidation state +III (Seifert et al., 2004; Schiller et al., 2005) used successfully for radiolabelling of interesting molecules, 188Re(V) complexes with bidentate or tetradentate ligands are still possible candidates for this approach. Complexes of 188Re(V) with DMSA (dimercapto succinic acid) and its derivatives or with ‘‘N2S2’’-, ‘‘N3S’’- or ‘‘N4’’-ligands like ECD (L,L-ethylene dicysteine diethyl ester), MAG3 (mercapto acetyl triglycine), and polyamines are commonly synthesised by a one-step route using huge amounts of auxiliary ligands (oxalic acid, cyclodextrines, citric acid, tartaric acid, ethylene diamine tetraacetic acid), reducing agents and in most cases also high temperatures. Another way to prepare the desired 188Re(V) complexes is a two-step procedure, in which, for example, a glucoheptonate, gluconate or citrate complex is first formed, and then in a second step undergoes a ligand exchange reaction with the appropriate ligand. Our work on the preparation of 99mTc and 188Re ‘4+1’ complexes has shown that the EDTA complexes of both radionuclides are suitable precursors for the formation of M(III)–mixed ligand complexes (Seifert et al., 2004). The preparation of 99mTc(III)–EDTA as well as 188 Re(III)–EDTA works well at room temperature and in volumes of 1–10 ml. While the reduction of pertechnetate succeeds in neutral solution, the formation of 188 Re(III)–EDTA requires strongly acidic conditions. Both complexes are stable to only a limited extent; especially 188 Re(III)–EDTA is re-oxidised to perrhenate in neutral solutions. Considering this distinctive re-oxidation behaviour of 188Re(III)–EDTA it should be possible to prepare 188 Re complexes of higher oxidation states by a combined ligand exchange/re-oxidation reaction of 188Re(III)aminopolycarboxylates with suitable ligands. This work presents a simple method for the preparation of oxorhenium(V) complexes as [ReO(ECD)] or [ReO(DMSA)2] and dioxorhenium(V) complexes as [ReO2(en)2]+, [ReO2(tau)]+ or [ReO2(cyclam)]+ (enethylene diamine, tau ¼ 1,4,8,11-tetraazaundecane, cyclam ¼ 1,4,8,11-tetraazacyclotetradecane) using the precursor complex 188Re(III)–EDTA.
purification. 99mTcO 4 was eluted from a commercial Mo/99mTc generator (Mallinckrodt, Petten, The Neth188 erlands). 188ReO W/188Re 4 was eluted from an generator (Oak Ridge National Laboratories, Oak Ridge, TN). ECD was synthesised by our group following a published procedure of Blondeau et al. (1967). The borohydride exchange resin (BER) was prepared by the method of Park et al. (2004). Chlorideform resin (Amberlites ion exchange resin, 12.5 g) was slurry-packed with water into a 30 ml fritted glass funnel mounted on a filter flask. Then, an aqueous sodium tetrahydroborate solution (200 ml, 0.25 M) was slowly passed through the resin over a period of 30 min. The resulting resins were washed thoroughly with distilled water until free of excess, and finally with ethanol. The tetrahydroborate-form anion exchange resin was then partially air-dried by removing ethanol on the surface of the BER. This resin was analysed for its tetrahydroborate content by measuring hydrogen evolution after acidification with 0.08 M HCl; the average capacity of BER was found to be 2.5 mequiv. of tetrahydroborate ion per gram. Thin-layer chromatography (TLC), high performance liquid chromatography (HPLC), and electrophoresis analyses were performed for controlling the identity, radiochemical purity, and stability of the preparations. For TLC studies silica gel strips (Kieselgel 60, Merck, Darmstadt, Germany) were used and developed with mobile phases of acetone and water, a mixture of nbutanol/acetic acid/water (3/2/3) or ethyl acetate. The strips were scanned with a raytest Rita radio analyzer (raytest IsotopenmeXgera¨te GmbH, Straubenhardt, Germany). The HPLC analyses were performed on a Perkin Elmer device consisting of a Turbo LC System with a quaternary pump (Series 200 LC Pump), a Programmable Absorbance Detector Model 785A (Perkin Elmer Instruments GmbH, Rodgau-Ju¨gesheim, Germany) and a home-made g-ray detector (well-type NaI(Tl) crystal). The HPLC analyses were carried out with a PRP-1 column (Hamilton, 250 mm 4 mm) using a gradient eluant of acetonitrile (A) with 0.1% TFA/ water (B) with 0.1% TFA, gradient elution: 0–10 min 0 to 50% A, 10–15 min 50% A, 15–18 min 50–100% A and a flow rate of 1.0 or 2.0 ml/min. The effluent from the column was monitored for the 99mTc and 188Re complexes by g-ray detection. Paper electrophoresis studies were performed at 300 V in 0.01 M phosphate buffer solution of pH 7.4 or with mixtures of phosphate buffer and acetonitrile as electrolytes.
2. Experimental
2.2. Radiolabelling
2.1. Materials and methods All solvents and other commercially available substances were of reagent grade and used without further
99
188
Re(III)–EDTA was prepared by adding 3–5 ml perrhenate eluate from a 188W/188Re generator together with 0.05 ml 1 N HCl to a kit vial containing 5 mg EDTA, 5 mg mannitol and 1.0 mg SnCl2 in freeze-dried
ARTICLE IN PRESS S. Seifert, H.-J. Pietzsch / Applied Radiation and Isotopes 64 (2006) 223–227
The re-oxidation studies performed with bidentate and tetradentate ligands resulted in high yields of the desired 188Re(V)-oxocomplexes when the ligands DMSA or ECD were dissolved in 0.1 M phosphate buffer pH 7.4 and added, with the exclusion of air, to the acidic 188 Re(III)–EDTA solution (Table 1). Thus, the ligand exchange reaction between 188Re(III)–EDTA, which was prepared under a nitrogen atmosphere in the kit vial, and the respective ligand occurs in neutral solution and only small amounts of perrhenate are formed. However, when the preparations were performed without the exclusion of air and after neutralisation of the 188 Re(III)–EDTA solution, 20–30% perrhenate was always detected in the final solution. Obviously, the neutralisation of the 188Re(III)-EDTA solution must be carried out in the presence of the appropriate ligand in order to prevent a fast re-oxidation of 188Re(III)–EDTA to perrhenate. The identity of the prepared 188Re complexes was confirmed by simultaneously prepared and well-characterised 99mTc complexes and cold rhenium complexes. 188 Re(V)–DMSA prepared from 188Re(III)–EDTA at room temperature was compared by HPLC with ‘‘kit-prepared’’ 99mTc(V)–DMSA and the 188Re-ECD preparation was identified in the same way (Fig. 1). The cationic dioxocomplexes with acyclic and cyclic polyamines were prepared according to the same procedure. The preparation of the complexes [188ReO2(en)2]+ and [188ReO2(tau)]+ also succeeded in high yields of 90–95% without addition of phosphate buffer solution at pH 9–10. It was observed, however,
form under nitrogen. After 20–30 min at room temperature the complex formation was finished; yield: 495% (TLC). Alternatively, the same complex was prepared using BER instead of SnCl2 as the reducing agent. For this purpose a mixture of 5 mg EDTA and 5 mg mannitol dissolved in 0.5 ml saline, and 2 ml perrhenate eluate containing 5 ml of 85% H3PO4, was added to a vial containing 10 mg BER under nitrogen. After 25 min reaction time at room temperature 90–95% of the desired complex was formed. For preparation of the 188Re(V) complexes 0.1–10 mg of the appropriate ligand dissolved in 1.0 ml of 0.1 M phosphate buffer solution of pH 7.4 was added under nitrogen flushing to 1.0 ml of the acidic precursor solution and the final solution was allowed to stand for 10–20 minutes at room temperature. In most cases an ultrasonic bath was used for incubations.
3. Results and discussion We found that [188Re]perrhenate is easily reduced to a Re(III)-complex without heating and with only 1 mg of stannous chloride in an acidic solution of EDTA, also in volumes of 10 ml. To avoid the undesired formation of stannous hydroxides, SnCl2 may be substituted by BER. Using optimised conditions the resulting 188 Re(III)–EDTA solution exhibits the same radiochemical purity, about 95%, as that determined for a Sn(II)-reduced solution. Moreover, the BER is easy to remove from the complex solution. 188
Table 1 Yields and reaction conditions for the preparation of
225
188
Re(V) complexes from
188
Re-EDTA
Complex formed
Yield [%]
Optimum reaction conditions
[188ReO(ECD)] [188ReO(DMSA)2] [188ReO2(en)2]+ [188ReO2(tau)]+ [188ReO2(cyclam)]+
9572 9473 9372 9472 5075
0.5 mg ligand, ultrasonic, 10 min, pH 7, N2 0.5 mg ligand, ultrasonic, 10 min, pH 7, N2 10 mg ligand, ultrasonic, 10 min, pH 10 5 mg ligand, ultrasonic, 10 min, pH 10 20 mg ligand, ultrasonic, 10 min, pH 10, N2
188
99m
Re(V)-DMSA
0
2
4
6 8 10 Time (min)
Tc(V)-DMSA
12
14
16
0
2
4
6 8 10 Time (min)
12
14
16
Fig. 1. Comparison of HPLC analyses of 188Re(V)–DMSA and a kit preparation of 99mTc(V)–DMSA; PRP-1 column; acetonitrile/ 0.1% TFA (A)//water/0.1% TFA (B); all three stereoisomers are well separated using a linear gradient 0–50% A in 20 min (tR values between 8 and 13 min).
ARTICLE IN PRESS
Intensity [Counts]
8000 7000 6000 5000 4000 3000 2000 1000 0
Intensity [Counts]
S. Seifert, H.-J. Pietzsch / Applied Radiation and Isotopes 64 (2006) 223–227
226
−
+
−
+
4000 3000 2000 1000 0
0
100 Position [mm]
200
0
Fig. 2. Paper electrophoresis analysis of [188ReO2(tua)]+ prepared from exchange purification (300 V, 45 min, 0.01 M phosphate buffer pH 7.4).
that the 188Re(III)–EDTA precursor complex only formed about 50% of the cyclam complex, despite high ligand excess and different reaction conditions (room temperature or 50–100 1C, ultrasonic, pH 6–10). In contrast to the reactions with ECD, DMSA or the acyclic amines en and tau, which resulted in high yields of the desired ligand exchange product, 50% or more of the starting activity was re-oxidised to perrhenate. This agrees with observations made by Prakash et al. (1996), who found that only complexes with acyclic polyamine ligands are formed by direct reduction of perrhenate in aqueous solutions, while the synthesis of Re-188 complexes with cyclic polyamines like cyclam using the same procedure failed. Obviously, the formation of the [188ReO2(cyclam)]+ complex needs longer time and higher activation energies than those needed for acyclic amine ligands. Thus, the re-oxidation to perrhenate is favoured against the ligand exchange reaction. The complexes were additionally characterised by paper electrophoresis (Fig. 2) and anion exchange separation using minicolumns filled with Dowex (1 8) ion exchange resin (Cl-form). The cationic complexes were eluted with water from the anion exchange column as radiochemically pure products, while the anionic impurities 188Re(III)–EDTA and 188ReO 4 were retained at the column. Attempts to accelerate the ligand exchange reaction and to improve the yields by using other 188Reaminopolycarboxylates were not successful. Only low labelling yields of 10–40% were achieved with weaker chelating agents like ethylene diamine diacetic acid or imino diacetic acid, despite of the use of 20 or 30 mg of the ligands and more stannous chloride. Surprisingly, the formation of the 188Re-diethylene triamine pentaacetic acid complex using the same mild conditions as for EDTA preparations resulted in unsatisfactory yields of only 65–70%.
4. Conclusions Rhenium-188(V) oxo- and dioxocomplexes with bidentate or tetradentate ligands were prepared in high
100 Position [mm]
200
188
Re(III)–EDTA; left: original sample, right: after anion
radiochemical yields (with exception of [188ReO2(cyclam)]+) by a combined re-oxidation/ligand exchange reaction starting from 188Re(III)–EDTA. The formation of the precursor complex and the subsequent preparation of the desired Re(V)-complexes may be performed under mild conditions (room temperature, neutral or weak basic solutions, low amounts of reducing agent, ligands, and auxiliary ligands) and allows consequently the labelling of sensitive biomolecules coupled to such types of chelates.
References Alberto, R., Schibli, R., Waibel, R., et al., 1999. Basic aqueous chemistry of [M(OH2)3(CO)3]+ (M ¼ Re, Tc) directed towards radiopharmaceutical application. Coord. Chem. Rev. 190–192, 901–919. Bla¨uenstein, P., 1990. Rhenium in nuclear medicine: general aspects and future goals. New J. Chem. 14, 405–407. Blondeau, P., Berse, C., Gravel, D., 1967. Dimerization of an intermediate during the sodium in liquid ammonium reduction of L-thiazolidine-4-carboxylic acid. Can. J. Chem. 45, 49–59. Blower, P.J., Lam, A., Knapp Jr., F.F., et al., 1996. Preparation, biodistribution and dosimetry of 188Re(V)DMSA in patients with disseminated bone metastases. Nucl. Med. Commun. 17, 258. Dadachova, E., Mirzadeh, S., 1997. The role of tin in the direct labelling of proteins with rhenium-188. Nucl. Med. Biol. 24, 605–608. Hsieh, B.-T., Hsieh, J.-F., Tsai, S.-C., et al., 1999. Rhenium188-labeled DTPA: a new radiopharmaceutical for intravascular radiation therapy. Nucl. Med. Biol. 26, 967–972. Knapp Jr., F.F., Callahan, A.P., Beets, A.L., et al., 1994. Processing of reactor-produced 188W for fabrication of clinical scale alumina-based 188W/188Re generators. Appl. Radiat. Isot. 45, 1123–1128. Lin, W.-Y., Hsieh, J.-F., Lin, C.-P., et al., 1999. Effect of reaction conditions on preparations of rhenium-188 hydroxyethylidene diphosphonate complexes. Nucl. Med. Biol. 26, 455–459. Mitterhauser, M., Wadsak, W., Eidherr, H., et al., 2004. Labelling of EDTMP (Multibone) with [111In], [99mTc] and
ARTICLE IN PRESS S. Seifert, H.-J. Pietzsch / Applied Radiation and Isotopes 64 (2006) 223–227 [188Re] using different carriers for ‘‘cross complexation’’. Appl. Radiat. Isot. 60, 653–658. Oh, S.J., Moon, D.H., Lee, W.W., et al., 2003. Automated preparation of 188Re-labeled radiopharmaceuticals for endovascular radiation therapy. Appl. Radiat. Isot. 59, 225–230. Park, S.H., Gwon, H.J., Park, K.B., 2004. Synthesis and characterization of 99mTc-labelled biologically active molecules using borohydride exchange resin as a reducing agent for the preparation of radiopharmaceuticals. J. Label. Compd. Radiopharm. 47, 683–697. Prakash, S., Went, M.J., Blower, P.J., 1996. Cyclic and acyclic polyamines as chelators of rhenium-186 and rhenium-188 for therapeutic use. Nucl. Med. Biol. 23, 543–549.
227
Schibli, R., Schwarzbach, R., Alberto, R., et al., 2002. Steps toward high specific activity labeling of biomolecules for therapeutic application: preparation of precursor [188Re(H2O)3(CO)3]+ and synthesis of tailor-made bifunctional ligand systems. Bioconjugate Chem. 13, 750–756. Schiller, E., Seifert, S., Tisato, F., et al., 2005. NS3/P (‘4+1’) coordination: relation of structure with antioxidation stability. Bioconjugate Chem. 16, 634–643. Seifert, S., Ku¨nstler, J.-U., Pietzsch, H.-J., et al., 2004. Novel procedures for preparing 99mTc(III) complexes with tetradentate/monodentate coordination of varying lipophilicity and adaptation to 188Re analogues. Bioconjugate Chem. 15, 856–863.
Applied Radiation and Isotopes 64 (2006) 223–227 www.elsevier.com/locate/apradiso
The 188Re(III)–EDTA complex—a multipurpose starting material for the preparation of relevant 188Re complexes under mild conditions S. Seifert, H.-J. Pietzsch Forschungszentrum Rossendorf, Institut fu¨r Bioanorganische und Radiopharmazeutische Chemie, Postfach 510 119, D-01314 Dresden, Germany Received 25 July 2005; received in revised form 17 August 2005; accepted 20 August 2005
Abstract An easy and gentle method for the preparation of 188Re(V) complexes with bidentate and tetradentate ligands is described starting from the precursor complex 188Re(III)–EDTA. That complex is prepared at room temperature in acidic solution and reacts by a combined re-oxidation/ligand exchange reaction with appropriate ligands like DMSA or ECD (DMSA ¼ dimercapto succinic acid, ECD ¼ L,L-ethylene dicysteine diethyl ester) or en, tau, and cyclam (en ¼ ethylene diamine, tau ¼ 1,4,8,11-tetraazaundecane, cyclam ¼ 1,4,8,11-tetraazacyclo-tetradecane) to the 188 Re(V)-oxo- and dioxocomplexes, respectively. The chelates were unambiguously identified by chromatographic comparison with spectroscopically characterised samples or known 99mTc-kit reconstitutions. The reaction succeeds under mild conditions (room temperature, short time, neutral or weak basic solutions) with high yields and has potential for labelling of sensitive biomolecules with 188Re. r 2005 Elsevier Ltd. All rights reserved. Keywords:
188
Re(V) complexes; Ligands; Re-oxidation
1. Introduction Rhenium-188 belongs to the most favourable radionuclides offering potential for targeted radiotherapy of cancer. It is currently obtained from the parent nuclide tungsten-188 through a generator system (Knapp et al., 1994). Due to its easy availability and suitable nuclear properties (Ebmax ¼ 2.1 MeV, t1/2 ¼ 16.9 h), 188Re is an attractive isotope for developing therapeutic radiopharmaceuticals (Bla¨uenstein, 1990; Blower et al., 1996; Prakash et al., 1996; Lin et al., 1999). The Corresponding author. Tel.: +49 351 2602442; fax: +49 351 2603232. E-mail address: [email protected] (S. Seifert).
associated g-emission (Eg ¼ 155 keV) could be additionally utilised for imaging and dosimetric purposes. Generally, there is a wide chemical analogy between 188 Re and 99mTc. Differences are observed in the kinetics of ligand exchange reactions and the redox behaviour. Especially the reduction of the perrhenate generator eluate in various ligand solutions needs more drastic conditions than those used for pertechnetate eluate. Normally, milligram amounts of stannous chloride or other reducing agents have to be used for complete reduction, e. g., of perrhenate to Re(V)-oxocomplexes with ‘‘N2S2’’- or ‘‘N3S’’-ligands, in contrast to the microgram amounts, necessary for the reduction of pertechnetate (Dadachova and Mirzadeh, 1997; Oh et al., 2003). Moreover, the complex formation only
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succeeds in limited volumes and in the majority of cases at high temperatures and under strongly acidic conditions (Mitterhauser et al., 2004; Hsieh et al., 1999). All these facts restrain the labelling of sensitive biomolecules according to the bifunctional approach. Besides the low valent organometallic tricarbonyl complexes (Alberto et al., 1999; Schibli et al., 2002) and the ‘4+1’ complexes at oxidation state +III (Seifert et al., 2004; Schiller et al., 2005) used successfully for radiolabelling of interesting molecules, 188Re(V) complexes with bidentate or tetradentate ligands are still possible candidates for this approach. Complexes of 188Re(V) with DMSA (dimercapto succinic acid) and its derivatives or with ‘‘N2S2’’-, ‘‘N3S’’- or ‘‘N4’’-ligands like ECD (L,L-ethylene dicysteine diethyl ester), MAG3 (mercapto acetyl triglycine), and polyamines are commonly synthesised by a one-step route using huge amounts of auxiliary ligands (oxalic acid, cyclodextrines, citric acid, tartaric acid, ethylene diamine tetraacetic acid), reducing agents and in most cases also high temperatures. Another way to prepare the desired 188Re(V) complexes is a two-step procedure, in which, for example, a glucoheptonate, gluconate or citrate complex is first formed, and then in a second step undergoes a ligand exchange reaction with the appropriate ligand. Our work on the preparation of 99mTc and 188Re ‘4+1’ complexes has shown that the EDTA complexes of both radionuclides are suitable precursors for the formation of M(III)–mixed ligand complexes (Seifert et al., 2004). The preparation of 99mTc(III)–EDTA as well as 188 Re(III)–EDTA works well at room temperature and in volumes of 1–10 ml. While the reduction of pertechnetate succeeds in neutral solution, the formation of 188 Re(III)–EDTA requires strongly acidic conditions. Both complexes are stable to only a limited extent; especially 188 Re(III)–EDTA is re-oxidised to perrhenate in neutral solutions. Considering this distinctive re-oxidation behaviour of 188Re(III)–EDTA it should be possible to prepare 188 Re complexes of higher oxidation states by a combined ligand exchange/re-oxidation reaction of 188Re(III)aminopolycarboxylates with suitable ligands. This work presents a simple method for the preparation of oxorhenium(V) complexes as [ReO(ECD)] or [ReO(DMSA)2] and dioxorhenium(V) complexes as [ReO2(en)2]+, [ReO2(tau)]+ or [ReO2(cyclam)]+ (enethylene diamine, tau ¼ 1,4,8,11-tetraazaundecane, cyclam ¼ 1,4,8,11-tetraazacyclotetradecane) using the precursor complex 188Re(III)–EDTA.
purification. 99mTcO 4 was eluted from a commercial Mo/99mTc generator (Mallinckrodt, Petten, The Neth188 erlands). 188ReO W/188Re 4 was eluted from an generator (Oak Ridge National Laboratories, Oak Ridge, TN). ECD was synthesised by our group following a published procedure of Blondeau et al. (1967). The borohydride exchange resin (BER) was prepared by the method of Park et al. (2004). Chlorideform resin (Amberlites ion exchange resin, 12.5 g) was slurry-packed with water into a 30 ml fritted glass funnel mounted on a filter flask. Then, an aqueous sodium tetrahydroborate solution (200 ml, 0.25 M) was slowly passed through the resin over a period of 30 min. The resulting resins were washed thoroughly with distilled water until free of excess, and finally with ethanol. The tetrahydroborate-form anion exchange resin was then partially air-dried by removing ethanol on the surface of the BER. This resin was analysed for its tetrahydroborate content by measuring hydrogen evolution after acidification with 0.08 M HCl; the average capacity of BER was found to be 2.5 mequiv. of tetrahydroborate ion per gram. Thin-layer chromatography (TLC), high performance liquid chromatography (HPLC), and electrophoresis analyses were performed for controlling the identity, radiochemical purity, and stability of the preparations. For TLC studies silica gel strips (Kieselgel 60, Merck, Darmstadt, Germany) were used and developed with mobile phases of acetone and water, a mixture of nbutanol/acetic acid/water (3/2/3) or ethyl acetate. The strips were scanned with a raytest Rita radio analyzer (raytest IsotopenmeXgera¨te GmbH, Straubenhardt, Germany). The HPLC analyses were performed on a Perkin Elmer device consisting of a Turbo LC System with a quaternary pump (Series 200 LC Pump), a Programmable Absorbance Detector Model 785A (Perkin Elmer Instruments GmbH, Rodgau-Ju¨gesheim, Germany) and a home-made g-ray detector (well-type NaI(Tl) crystal). The HPLC analyses were carried out with a PRP-1 column (Hamilton, 250 mm 4 mm) using a gradient eluant of acetonitrile (A) with 0.1% TFA/ water (B) with 0.1% TFA, gradient elution: 0–10 min 0 to 50% A, 10–15 min 50% A, 15–18 min 50–100% A and a flow rate of 1.0 or 2.0 ml/min. The effluent from the column was monitored for the 99mTc and 188Re complexes by g-ray detection. Paper electrophoresis studies were performed at 300 V in 0.01 M phosphate buffer solution of pH 7.4 or with mixtures of phosphate buffer and acetonitrile as electrolytes.
2. Experimental
2.2. Radiolabelling
2.1. Materials and methods All solvents and other commercially available substances were of reagent grade and used without further
99
188
Re(III)–EDTA was prepared by adding 3–5 ml perrhenate eluate from a 188W/188Re generator together with 0.05 ml 1 N HCl to a kit vial containing 5 mg EDTA, 5 mg mannitol and 1.0 mg SnCl2 in freeze-dried
ARTICLE IN PRESS S. Seifert, H.-J. Pietzsch / Applied Radiation and Isotopes 64 (2006) 223–227
The re-oxidation studies performed with bidentate and tetradentate ligands resulted in high yields of the desired 188Re(V)-oxocomplexes when the ligands DMSA or ECD were dissolved in 0.1 M phosphate buffer pH 7.4 and added, with the exclusion of air, to the acidic 188 Re(III)–EDTA solution (Table 1). Thus, the ligand exchange reaction between 188Re(III)–EDTA, which was prepared under a nitrogen atmosphere in the kit vial, and the respective ligand occurs in neutral solution and only small amounts of perrhenate are formed. However, when the preparations were performed without the exclusion of air and after neutralisation of the 188 Re(III)–EDTA solution, 20–30% perrhenate was always detected in the final solution. Obviously, the neutralisation of the 188Re(III)-EDTA solution must be carried out in the presence of the appropriate ligand in order to prevent a fast re-oxidation of 188Re(III)–EDTA to perrhenate. The identity of the prepared 188Re complexes was confirmed by simultaneously prepared and well-characterised 99mTc complexes and cold rhenium complexes. 188 Re(V)–DMSA prepared from 188Re(III)–EDTA at room temperature was compared by HPLC with ‘‘kit-prepared’’ 99mTc(V)–DMSA and the 188Re-ECD preparation was identified in the same way (Fig. 1). The cationic dioxocomplexes with acyclic and cyclic polyamines were prepared according to the same procedure. The preparation of the complexes [188ReO2(en)2]+ and [188ReO2(tau)]+ also succeeded in high yields of 90–95% without addition of phosphate buffer solution at pH 9–10. It was observed, however,
form under nitrogen. After 20–30 min at room temperature the complex formation was finished; yield: 495% (TLC). Alternatively, the same complex was prepared using BER instead of SnCl2 as the reducing agent. For this purpose a mixture of 5 mg EDTA and 5 mg mannitol dissolved in 0.5 ml saline, and 2 ml perrhenate eluate containing 5 ml of 85% H3PO4, was added to a vial containing 10 mg BER under nitrogen. After 25 min reaction time at room temperature 90–95% of the desired complex was formed. For preparation of the 188Re(V) complexes 0.1–10 mg of the appropriate ligand dissolved in 1.0 ml of 0.1 M phosphate buffer solution of pH 7.4 was added under nitrogen flushing to 1.0 ml of the acidic precursor solution and the final solution was allowed to stand for 10–20 minutes at room temperature. In most cases an ultrasonic bath was used for incubations.
3. Results and discussion We found that [188Re]perrhenate is easily reduced to a Re(III)-complex without heating and with only 1 mg of stannous chloride in an acidic solution of EDTA, also in volumes of 10 ml. To avoid the undesired formation of stannous hydroxides, SnCl2 may be substituted by BER. Using optimised conditions the resulting 188 Re(III)–EDTA solution exhibits the same radiochemical purity, about 95%, as that determined for a Sn(II)-reduced solution. Moreover, the BER is easy to remove from the complex solution. 188
Table 1 Yields and reaction conditions for the preparation of
225
188
Re(V) complexes from
188
Re-EDTA
Complex formed
Yield [%]
Optimum reaction conditions
[188ReO(ECD)] [188ReO(DMSA)2] [188ReO2(en)2]+ [188ReO2(tau)]+ [188ReO2(cyclam)]+
9572 9473 9372 9472 5075
0.5 mg ligand, ultrasonic, 10 min, pH 7, N2 0.5 mg ligand, ultrasonic, 10 min, pH 7, N2 10 mg ligand, ultrasonic, 10 min, pH 10 5 mg ligand, ultrasonic, 10 min, pH 10 20 mg ligand, ultrasonic, 10 min, pH 10, N2
188
99m
Re(V)-DMSA
0
2
4
6 8 10 Time (min)
Tc(V)-DMSA
12
14
16
0
2
4
6 8 10 Time (min)
12
14
16
Fig. 1. Comparison of HPLC analyses of 188Re(V)–DMSA and a kit preparation of 99mTc(V)–DMSA; PRP-1 column; acetonitrile/ 0.1% TFA (A)//water/0.1% TFA (B); all three stereoisomers are well separated using a linear gradient 0–50% A in 20 min (tR values between 8 and 13 min).
ARTICLE IN PRESS
Intensity [Counts]
8000 7000 6000 5000 4000 3000 2000 1000 0
Intensity [Counts]
S. Seifert, H.-J. Pietzsch / Applied Radiation and Isotopes 64 (2006) 223–227
226
−
+
−
+
4000 3000 2000 1000 0
0
100 Position [mm]
200
0
Fig. 2. Paper electrophoresis analysis of [188ReO2(tua)]+ prepared from exchange purification (300 V, 45 min, 0.01 M phosphate buffer pH 7.4).
that the 188Re(III)–EDTA precursor complex only formed about 50% of the cyclam complex, despite high ligand excess and different reaction conditions (room temperature or 50–100 1C, ultrasonic, pH 6–10). In contrast to the reactions with ECD, DMSA or the acyclic amines en and tau, which resulted in high yields of the desired ligand exchange product, 50% or more of the starting activity was re-oxidised to perrhenate. This agrees with observations made by Prakash et al. (1996), who found that only complexes with acyclic polyamine ligands are formed by direct reduction of perrhenate in aqueous solutions, while the synthesis of Re-188 complexes with cyclic polyamines like cyclam using the same procedure failed. Obviously, the formation of the [188ReO2(cyclam)]+ complex needs longer time and higher activation energies than those needed for acyclic amine ligands. Thus, the re-oxidation to perrhenate is favoured against the ligand exchange reaction. The complexes were additionally characterised by paper electrophoresis (Fig. 2) and anion exchange separation using minicolumns filled with Dowex (1 8) ion exchange resin (Cl-form). The cationic complexes were eluted with water from the anion exchange column as radiochemically pure products, while the anionic impurities 188Re(III)–EDTA and 188ReO 4 were retained at the column. Attempts to accelerate the ligand exchange reaction and to improve the yields by using other 188Reaminopolycarboxylates were not successful. Only low labelling yields of 10–40% were achieved with weaker chelating agents like ethylene diamine diacetic acid or imino diacetic acid, despite of the use of 20 or 30 mg of the ligands and more stannous chloride. Surprisingly, the formation of the 188Re-diethylene triamine pentaacetic acid complex using the same mild conditions as for EDTA preparations resulted in unsatisfactory yields of only 65–70%.
4. Conclusions Rhenium-188(V) oxo- and dioxocomplexes with bidentate or tetradentate ligands were prepared in high
100 Position [mm]
200
188
Re(III)–EDTA; left: original sample, right: after anion
radiochemical yields (with exception of [188ReO2(cyclam)]+) by a combined re-oxidation/ligand exchange reaction starting from 188Re(III)–EDTA. The formation of the precursor complex and the subsequent preparation of the desired Re(V)-complexes may be performed under mild conditions (room temperature, neutral or weak basic solutions, low amounts of reducing agent, ligands, and auxiliary ligands) and allows consequently the labelling of sensitive biomolecules coupled to such types of chelates.
References Alberto, R., Schibli, R., Waibel, R., et al., 1999. Basic aqueous chemistry of [M(OH2)3(CO)3]+ (M ¼ Re, Tc) directed towards radiopharmaceutical application. Coord. Chem. Rev. 190–192, 901–919. Bla¨uenstein, P., 1990. Rhenium in nuclear medicine: general aspects and future goals. New J. Chem. 14, 405–407. Blondeau, P., Berse, C., Gravel, D., 1967. Dimerization of an intermediate during the sodium in liquid ammonium reduction of L-thiazolidine-4-carboxylic acid. Can. J. Chem. 45, 49–59. Blower, P.J., Lam, A., Knapp Jr., F.F., et al., 1996. Preparation, biodistribution and dosimetry of 188Re(V)DMSA in patients with disseminated bone metastases. Nucl. Med. Commun. 17, 258. Dadachova, E., Mirzadeh, S., 1997. The role of tin in the direct labelling of proteins with rhenium-188. Nucl. Med. Biol. 24, 605–608. Hsieh, B.-T., Hsieh, J.-F., Tsai, S.-C., et al., 1999. Rhenium188-labeled DTPA: a new radiopharmaceutical for intravascular radiation therapy. Nucl. Med. Biol. 26, 967–972. Knapp Jr., F.F., Callahan, A.P., Beets, A.L., et al., 1994. Processing of reactor-produced 188W for fabrication of clinical scale alumina-based 188W/188Re generators. Appl. Radiat. Isot. 45, 1123–1128. Lin, W.-Y., Hsieh, J.-F., Lin, C.-P., et al., 1999. Effect of reaction conditions on preparations of rhenium-188 hydroxyethylidene diphosphonate complexes. Nucl. Med. Biol. 26, 455–459. Mitterhauser, M., Wadsak, W., Eidherr, H., et al., 2004. Labelling of EDTMP (Multibone) with [111In], [99mTc] and
ARTICLE IN PRESS S. Seifert, H.-J. Pietzsch / Applied Radiation and Isotopes 64 (2006) 223–227 [188Re] using different carriers for ‘‘cross complexation’’. Appl. Radiat. Isot. 60, 653–658. Oh, S.J., Moon, D.H., Lee, W.W., et al., 2003. Automated preparation of 188Re-labeled radiopharmaceuticals for endovascular radiation therapy. Appl. Radiat. Isot. 59, 225–230. Park, S.H., Gwon, H.J., Park, K.B., 2004. Synthesis and characterization of 99mTc-labelled biologically active molecules using borohydride exchange resin as a reducing agent for the preparation of radiopharmaceuticals. J. Label. Compd. Radiopharm. 47, 683–697. Prakash, S., Went, M.J., Blower, P.J., 1996. Cyclic and acyclic polyamines as chelators of rhenium-186 and rhenium-188 for therapeutic use. Nucl. Med. Biol. 23, 543–549.
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Schibli, R., Schwarzbach, R., Alberto, R., et al., 2002. Steps toward high specific activity labeling of biomolecules for therapeutic application: preparation of precursor [188Re(H2O)3(CO)3]+ and synthesis of tailor-made bifunctional ligand systems. Bioconjugate Chem. 13, 750–756. Schiller, E., Seifert, S., Tisato, F., et al., 2005. NS3/P (‘4+1’) coordination: relation of structure with antioxidation stability. Bioconjugate Chem. 16, 634–643. Seifert, S., Ku¨nstler, J.-U., Pietzsch, H.-J., et al., 2004. Novel procedures for preparing 99mTc(III) complexes with tetradentate/monodentate coordination of varying lipophilicity and adaptation to 188Re analogues. Bioconjugate Chem. 15, 856–863.